Hepatitis C virus (HCV) is a major cause of chronic liver disease. We showed that HCV could be classified into 6 major genotypes. Prototype strains of the various genotypes have been biologically amplified in chimpanzees and distributed for use as challenge inocula in studies of passive and active immunoprophylaxis, etc. Full-length cDNA clones of genotypes 1a, 1b and 2a have been constructed and transcribed RNA used to transmit hepatitis C to chimpanzees by in vivo hepatic transfection. With these reagents we are pursuing studies of the immunopathogenesis of HCV infections in chimpanzees, a surrogate of man. Humoral immunity. We have shown that humoral immunity is not important in the control of acute infection, since neutralizing antibodies (NtAb), as measured against pseudotyped retroviruses bearing the HCV genotype 1a glycoproteins, were detected in only 1 of 4 animals with acute resolving 1a infection. High titer NtAb were detected in 2 of 4 chimpanzees with persistent infection. Similarly, high titers of NtAb were detected during persistent 1a infection in a patient with posttransfusion hepatitis. In general, NtAb responses correlated with humoral responses to the E1 and E2 proteins. We have developed infectious pseudo-typed particles bearing the envelope proteins of genotypes 1b, 2a, 3a, 4a, 5a and 6a and demonstrated that NtAb that develop during persistent 1a infection can cross-neutralize pseudo-particles of genotypes 1b, 4a, 5a and 6a, but have limited cross-reactivity against 2a and 3a. Cellular immunity. We have demonstrated that the intrahepatic T cell responses were vigorous in chimpanzees with viral control but their strength does not predict the final outcome of infection. Since animals infected with the same HCV strain had mild to severe acute liver disease these studies suggested that the host response is the principal determinant of the severity of acute HCV. Host gene expression. Gene expression analysis of HCV infected chimpanzees with different outcomes identified intrahepatic responses associated with viremia, such as interferon alpha, as well as outcome-specific responses associated with clearance, such as genes involved in the adaptive immune response. These studies also showed that acute infection influences the expression of many other genes, including those involved with metabolism, apoptosis and cell cycle regulation. Virus evolution. We have studied the correlation between host response, virus evolution and outcome in chimpanzees infected with different HCV strains. Changes in the polyprotein sequence are not selected until after the initial decrease in virus titers and until after the development of cellular immune responses and hepatitis. Subsequently mutations emerge repeatedly suggesting that mutations might represent an important mechanism for HCV persistence. However, the emergence of such variants does not necessarily lead to viral persistence. We have demonstrated that mutations that develop in animals with a monoclonal infection in some instances do represent CTL escape mutants. Protective immunity. We have demonstrated that sterilizing immunity can be achieved by repeated infection of chimpanzees, but that this immunity is strain-specific. This immunity is not mediated by NtAb, but is correlated with anamnestic T cell responses. However, immunity acquired during acute HCV infection does not necessarily prevent persistent infection, even following re-challenge with the homologous monoclonal virus. Our analyses suggest that the persisting viruses represent immune escape variants. In vivo functional analysis. The availability of infectious cDNA clones has permitted mutational analysis of HCV in vivo. We demonstrated that several regions of the 3' UTR were critical for replication of HCV. We also found that the E2 HVR1 is not critical for viability of HCV. Mutational analysis of the gene that encodes the p7 protein demonstrated that it is critical for the viability of HCV and contains critical genotype-specific sequences. We have determined the effect of mutations that permit replication of a subgenomic replicon derived from the HCV strain Con1 in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, in Huh-7 cells increased by introducing adaptive mutations in NS3 and NS5A. However, these adaptive mutations negatively influenced in vivo infectivity. In vitro mutagenesis. We have initiated mutational analysis of the HCV envelope glycoproteins using the pseudotype virus system and analyzing the putative HCV fusion peptide located in E1. We tested 20 mutants containing Ala and/or Pro substitutions at individual amino acid positions within this fusion peptide. We found that Ala substitutions at a number of conserved Leu, Gly or Cys positions significantly reduced the number of infected Huh-7 cells. Substitutions of these Leu, Gly, and Cys residues with Pro practically abolished infectivity. Vaccine development. A DNA vaccine expressing a truncated E2 protein generated high anti-E2 titers in chimpanzees, but it did not prevent infection following homologous challenge. Recent studies have demonstrated that neither animal had detectable NtAb against the homologous HCV pseudo-particles, which might explain why these animals were not protected. DNA vaccine vectors encoding other modified E2 proteins have been tested in rhesus monkeys. It remains to be determined whether they generated NtAb. Similar studies of modified surface-expressed forms of E1 have been performed. The E1 constructs were tested as DNA vaccines in mice and a strategy of DNA vaccination followed by a vaccinia-mediated protein boost was explored. However, the vaccines did not generate NtAb. Researchers at Chiron previously reported on 5 chimpanzees that were protected against homologous HCV-1 challenge after vaccination with purified envelope glycoproteins. They found that 4 of the 5 vaccinees never became viremic during follow-up. The fifth became infected but the infection was resolved. In a retrospective study, we found that all vaccinees displayed a significant NtAb response at the time of challenge as measured with retroviral pseudo-particles bearing the glycoproteins of another 1a strain (strain H77). The reciprocal NtAb titer was as high as 3200 and the sera efficiently cross-neutralized HCVpp 4a, 5a and 6a. In contrast, HCVpp 2a and 3a were minimally neutralized if at all. Thus, this study shows that it is possible to induce high titers of cross-reactive NtAb with recombinant HCV envelope glycoproteins. These results provide encouragement for the development of an effective recombinant vaccine against hepatitis C. Using the pseudo-typed viruses bearing the envelope proteins of the various genotypes of HCV we have demonstrated cross-reactivity of monoclonal antibodies derived from humans or chimpanzees. Such antibodies could potentially be used in passive immnunoprophylaxis. A surrogate model for HCV. We have constructed an infectious clone of GB virus B (GBV-B), a monkey virus that is the closest relative to HCV. Experimental infection with GBV-B results in acute viral hepatitis in tamarins. By testing deletion mutants of the GBV-B clone in tamarins we have shown that each of the predicted encoded GBV-B proteins is critical. However, viruses with deletions of specific domains within the 3' UTR were found to be viable. Infection with one such deletion mutant produced a persistent infection with chronic hepatitis. This observation strengthens the relevance of GBV-B as a surrogate model for the study of HCV. Most recently, we have demonstrated that a p7 like protein of GBV-B exists and that this protein (p13) most likely is twice as large as the equivalent protein of HCV. We have demonstrated that the p13 protein has two smaller subunits, but only the C-terminal subunit is essential for infectivity.